Phenolic Responses to Esca-Associated Fungi in Differently Decayed

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Phenolic responses to esca-associated fungi in differently decayed grapevine woods from different trunk parts of ‘Cabernet Sauvignon’ Denis Rusjan, Martina Persic, Matevž Likar, Katerina Biniari, and Maja Mikulic-Petkovsek J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02188 • Publication Date (Web): 10 Jul 2017 Downloaded from http://pubs.acs.org on July 11, 2017

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Journal of Agricultural and Food Chemistry

Phenolic responses to esca-associated fungi in differently decayed grapevine woods from different trunk parts of ‘Cabernet Sauvignon’

Denis Rusjan†1, Martina Persic†, Matevž Likar‡, Katerina Biniari+ and Maja Mikulic-Petkovsek†

†

University of Ljubljana, Biotechnical Faculty, Department of Agronomy, Chair for Fruit, Wine and Vegetable

Growing, Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia ‡

+

University of Ljubljana, Biotechnical Faculty, Department of Biology, Večna pot 111, SI-1000 Ljubljana Agricultural University of Athens, Faculty of Crop Science, Laboratory of Viticulture, Iera Odos 75, Athens,

Greece

1

Corresponding author (e-mail: [email protected], Fax: +386 1 423 10 88, Tel: +386 1 320 31 51)

ACS Paragon Plus Environment


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ABSTRACT

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Alterations in phenolic contents were studied in esca symptomatic (Sym) and

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asymptomatic (Asym) vines of ‘Cabernet Sauvignon’ based on wood conditions (healthy –

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HLT, necrotic – Nec and rotten – Rot) and vine parts (head, trunk and rootstock). In Asym

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vines, only Alternaria alternate was identified in Nec wood, while the HLT wood of Sym

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vines was colonised by Botryospaeriaceae sp. and Aureobasidium pullulans, Nec wood by

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Fomitiporia mediterranea and Rot wood by Fomitiporia mediterranea and Phaeomoniella

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chlamydospora. Esca infection caused a significant accumulation of gallic acid, total

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flavanols, stilbenes (STB) and of total analysed phenolics (TAP) in all studied woods,

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especially in Nec wood. In Asym vines, TAP in the head increased with necrosis but in

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Sym it decreased, while TAP in the trunk and rootstock of Sym showed an opposite

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response. The significantly highest contents of procyanidins (Pcys), catechin, epicatechin,

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epicatechin gallates and Pcys dimers and tetramers were measured in HLT wood in the

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head and in Nec wood in the trunk of Sym vines. The significant increase of STB content

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was not caused only by esca infection in HLT wood, but also by necrosis in Asym vines,

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especially of ε-viniferin glucoside, resveratrol glycosides and astringin. The obtained

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results suggest that the alteration in phenolics differed not only due to esca infection but

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also due to the wood conditions and vine part, which might reflect the impact of the

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duration of the presence of the pathogen in different parts of the vine.

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Keywords: grapevine trunk diseases, necrosis, Phaeomoniella chlamydospora,

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polymerisation, procyanidins, secondary metabolites

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2 Environment ACS Paragon Plus

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INTRODUCTION

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Grapevine trunk diseases (GTD), due to their expansion and complexity, have become

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a major economic problem in winegrowing regions all around the world, particularly in

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Europe in the 90s of the last century, when the use of fungicides based on arsenic was

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prohibited. Esca, a grapevine disease, also known as grapevine apoplexy, has been long

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recognised and is characterised by wood deterioration, discoloration and decay, which

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frequently leads to partial or entire vine collapse. 1-3 Esca disease is caused by infection of

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the vine with various fungi, of which the most frequently reported and studied are

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Phaeomoniella

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mediterranes and fungi from the Botryosphaeriaceace family.1, 4-6 Furthermore, appearance

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of the disease also depends on interactions among the pathogen, grapevine variety, climatic

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and soil characteristics, vine pruning, grafting and spraying etc.1, 7-9 After infection with

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fungi associated with Esca, disease symptoms do not appear on the vine immediately but

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usually take a few years, when certain biochemical, physiological, structural and

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morphological changes in different plant organs and tissues occur.3,10 However, nine

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months after inoculation of the ‘Sangiovese’ grapevine variety with Phaeomoniella

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chlamydospora, wood discoloration and colonization were observed at 47.1 cm and 40.3

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cm, respectively.1 Chronic esca syndromes inside the trunk can be described as (i) white

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rot converting hard wood into soft and spongy tissue, (ii) red to dark brown or black spots,

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usually visible around the annual growth section, (iii) brown to black streaks in the wood

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and (iv) longitudinal cracks in the wood.1,6-7 Plants usually respond to infection by the

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pathogen with the production of certain flavonoid compounds, as a spontaneous and

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natural defence against the pathogen.11-12

chlamydospora,

Phaeoacremonium

aleophilum


and

Fomitiporia


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In recent decades, major attention has been devoted to phenolic compounds, due to

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their antimicrobial and often antioxidative function in the sphere of infection.10,13 One of

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the first reports on polyphenols in deteriorated wood dates back to 1995, when found an

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increase of stilbenes content as a wood resistance response to brown and white rot fungi.14

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The presence of resveratrol and other ε-viniferins in brown-red esca infected wood has

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been already reported.15 This study was later upgraded by a more detailed study including

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several other stilbenes, which have a different response to esca infection.16 In in-vitro

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conditions, p-coumaric and caffeic acid, catechin, tannins and certain others phenolics

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inhibited enzyme activities involved in lignin degradation after infection with Petri disease;

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suggested that phenolics have a direct impact on fungal growth and their sporulation.13 A

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general increment of phenolics in symptomatic wood tissue of three different grapevine

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varieties infected by Phaeomoniella chlamydospora was reported, especially the impact of

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trans-resveratrol and ε-viniferin.17 In a further in-vitro experiment, it was shown that

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oligomers of hydroxystilbenoids significantly reduced the growth of strains of

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Botryosphaeriaceae associated fungi.10 Although studies on GTD have been ongoing for

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several decades, many hypotheses and questions still remain. To date, studies have been

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conducted on various vine organs, mainly leaves and berries, but some on wood, generally

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focusing on infected plants. However, according to the available scientific literature, a

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detailed comparison of phenolic compounds among various deteriorated woods between

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esca symptomatic and asymptomatic plants using HPLC-MS analysis has not yet been

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done.

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In this research, we studied the alteration of phenolics in various deteriorated woods

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from different parts of the vine, in esca symptomatic and asymptomatic vines of the highly

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sensitive grapevine ‘Cabernet Sauvignon’ (Vitis vinifera L.). The goal of the research was


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to determine specific responses of certain phenolic substances in different deteriorated

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woods as a natural defence mechanism of the grapevine to the esca disease. The present

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study gives insight into the complex characteristics of the biochemical pathway in GTD

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and partially contributes to the clarification of the esca phytopathology.

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MATERIALS AND METHODS

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Plant material. The plant material was sampled from 12-years old vines of the

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grapevine (Vitis vinifera L.) ‘Cabernet Sauvignon’, grafted on SO4 (Vitis berlandieri ×

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Vitis riparia) – the most susceptible vines to esca infection.2, 9 The vines were grown in a

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vineyard located in the Goriška brda winegrowing district (46°00’N, 13°05’S) of

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Primorska, western Slovenia. The vines were trained on double guyot and planted at

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distances 1.0 m x 2.2 m. The experimental vineyard is characterised by a sub-

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Mediterranean climate, at 115 m above sea level, 20% inclination, south-east exposed

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terraces and flysch soil type.

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During the growth season in 2015, the vines were marked according to visual typical

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esca as symptomatic (Sym – vines with tiger-chlorotic and necrotic spots on leaves, and/or

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partly dead canes, spurs) or asymptomatic (Asym – presumably healthy, esca-free plants;

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without any symptoms on leaves, canes or trunk) vines. Before vine sampling in the

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vineyard, the vines were tested with ELISA for the potential presence of the viruses GFLV,

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ArMV, GFkV, GVA, GLRaV-1 and GLRaV-3; they were shown to be virus-free. Five

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Sym and five Asym plants were sampled during the next winter (25th January 2016) for

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further study. They were removed from the vineyard, each vine was cut longitudinally to

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obtain a clear overview of the wood conditions in different vine parts: head (head – the

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upper and major part of the trunk), trunk (trunk – middle part from under the head to the



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graft site) and rootstock (rootstock – below the graft site) (Figure 1). Woods in different

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conditions - healthy (HLT - bright, healthy wood), necrotic (Nec - brownish, necrotic

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wood) and rotten (Rot - whitish, rotten wood) were sampled simultaneously in each

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particular part of Asym and Sym vines (Figure 1). It must be stressed that HLT and Nec

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wood was observed on head and trunk, and only HLT on rootstock with Asym vines, while

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with Sym vines HLT, Nec and Rot on head and trunk, and only HLT and Nec on rootstock.

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Rot wood was not observed on rootstock (Figure 1). After longitudinally cutting the vines,

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the entire surface of the vine was immediately cleaned with 80% ethanol. The surface at

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the sampling point was subsequently slightly removed with a scalpel and approx. 2 g of

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wood was collected with a drill (Φ 5 mm). Before and after each wood spot sampling, the

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drill was also cleaned with 80% ethanol. Wood was sampled from each of five vines per

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group (Sym and Asym), on the basis of the status and vine part; five replications per wood

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status and vine part were finally prepared from Sym and Asym vines. The obtained wood

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samples were collected into falcon tubes, immediately frozen in liquid nitrogen and stored

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at -80 °C for further analysis.

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Pathogen identification. For DNA extraction, plant material was obtained from

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grapevine wood by drilling. The wooden splints were ground into a fine powder using

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metal beads with a Bertin MiniLys® at 4000 rpm. The samples were frozen in liquid

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nitrogen for 2 min and then homogenised for 30 s. The cycle was repeated three times.

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DNA was extracted from 50 mg of homogenised plant material using a DNeasy Plant Mini

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Kit (Qiagen), following the modified manufacturer’s instructions.18

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The ITS rDNA region 5.8S-ITS2-28S segment of fungal rDNA operon was amplified,

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using primers ITS1F-ITS4.19-20 The PCR reaction mixture (25 µl) contained: 2.5 µl 10×

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PCR buffer, 2.5 mM MgCl2, 200 µM of each nucleotide, 500 nM of each primer, 0.75 U


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DNA polymerase and 12.5 µl of the 100-fold diluted template. PCR amplification was

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performed as follows: 94°C for 1 min, followed by 35 cycles of denaturation at 94°C for

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35 s, annealing at 55°C for 35 s and elongation at 72°C for 30 s. The elongation step was

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increased by 5 s every cycle. The final extension was at 72°C for 10 min.

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The amplified 18S rDNA regions were cloned prior to sequencing by ligation of the

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PCR products into the pGEM-T easy vector (Promega, USA). The ligation product was

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cloned into competent JM109 cells and cultured on LB agar containing X-Gal/ isopropyl-

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beta-D-thiogalactopyranoside and 50 µl/ml ampicillin (Sigma). Plasmid extraction was

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carried out from white colonies, and cloning was verified by colony PCR with T7 and SP6

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primers. Cycle-sequencing reactions were performed with T7 and SP6 primers using

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BigDye terminator Ready Reaction Cycle Sequencing kits on an ABI 3730xl DNA

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analyser (Applied Biosystems), as provided by the Macrogen Company (Korea).

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The sequences were subjected to GenBank searches, using the default option of

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gapped-BLAST,21 and aligned with the closest matches and additional representatives of

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groups from GenBank. Maximum-likelihood analysis was performed using MEGA7.22 The

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robustness of the internal branches was assayed by bootstrap analysis (1,000 runs). The

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evolutionary history was inferred by using the Maximum Likelihood method based on the

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Kimura 2-parameter model.23

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Phenolic extraction and quantification. Phenolic extraction from wood samples was

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performed according to the already reported method.24 Approximately 0.2 g (exact weight

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was recorded) of fine cooled wood powder was extracted into 2 mL MeOH for an hour in

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an ultrasonic bath. The obtained extracts were centrifuged at 10,000 rpm for 20 min, the

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supernatant was then filtered with a Chromafil AO-20/25 polyamide filter (Macherey-

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Nagel, Germany) and placed into vials, which were kept at -80 °C until analysis with the



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HPLC (high performance liquid chromatography) method. The analysis of phenolics was

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performed on the HPLC-DAD system; Thermo Finnigan Surveyor HPLC system (Thermo

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Scientific, San Jose, USA). Spectra of the compounds were recorded at 280 nm for flavan-

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3-ols and cinnamic acid derivatives, and at 350 nm for flavonols, ellagic acid derivatives

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and stilbenes. A Gemini C18 (150×4.6 mm 3 µm; Phenomenex, Torrance, USA) column

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was used, which operated at 25 °C. Elution solvents were (A) aqueous 0.1% formic acid in

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bidistilled water and (B) 0.1% formic acid in acetonitrile. The injection volume was 20 µl

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at a flow rate of 0.6 ml/min. Samples were eluted according to linear gradients.25

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For identification of individual phenolic compound, mass spectrometry (Thermo

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Scientific, LCQ Deca XP MAX) with electrospray ionization (ESI) operating in negative

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ion mode was carried out using full scan data-dependent MSn scanning from m/z 115 to

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1800. The injection volume was 10 µl, the flow rate was maintained at 0.6 ml min-1 and the

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capillary temperature was 250 °C. The obtained spectral data was elaborated using the

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Excalibur software (Thermo Scientific), the compounds were identified and confirmed by

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comparing a retention time, their spectra and by fragmentation (Table 1, Figure 2).

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However, we were unable to get information about the stereochemistry of a double bond.

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Contents of phenolics were evaluated from peak areas of the sample and the

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corresponding standards and expressed in µg/kg fresh weight (FW) of wood. For

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compounds lacking standards, quantification was carried out using similar compounds as

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standards; monogalloyled procyanidin dimer, procyanidin trimers, procyanidin tetramers

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were calculated from standard curve of procyanidin dimer; piceatannol was calculated

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from the standard curve of resveratrol; epicatechin gallate was calculated from the standard

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curve of epicatechin (Table 1).


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Statistical analysis. The analysis of data was carried out using Statgraphics Plus 4.0.

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To distinguish among contents of phenolic compounds, one-way analysis of variance and

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Duncan’s test were used. Values lower or equal to 0.05 were considered statistically

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significant. Results are presented as mean ± standard errors of five repetitions.

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RESULTS AND DISCUSSION

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Determined fungi. The sampled vines characterised as esca symptomatic (Sym) and

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asymptomatic (Asym), with various wood conditions (HLT, Nec and Rot), differed in the

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presence of esca associated fungi, as expected (Figure 3). Healthy (HLT) grapevine wood

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from the head of Asym was colonised only by Alternaria spp., with the highest similarity

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to species A. alternata (Figure 3), which can infect and cause disease in at least 380

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different plant species,26 and is usually present in decomposing soil and organic matter.27

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Alternaria spp. is fifth of the thirty most common fungi isolated from vine leaves, in which

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its presence reached 50% in Asym and less than 30% in esca Sym plants.6 Moreover, A.

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alternata produces metabolites that inhibit sporulation of grapevine downy mildew

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(Plasmopara viticola).28 There was a higher presence of esca associated fungi in Sym than

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in Asym plants, which largely coincides with previous reports1, 4-6. We can speculate that

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Nec wood in Asym vines is a result of annual winter and summer pruning, when the wood

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below wounds is subjected to stress conditions. In the HLT wood of Sym vines,

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Botryosphaeriaceae sp. were identified, the most frequent fungi on esca Asym and Sym

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leaves, in which the presence was 85% and 75%, respectively.6 Moreover, HLT wood of

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infected vines is colonized by Phaeomoniella chlamydospora (Pch) and Phaeoacremonium

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aleophilum (Pal),1 which was confirmed in our study for Pch only. On the other hand, we

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also identified a yeast-like fungus Aureobasidium pullulans (Ap) in HLT wood of Sym



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vines, which is a naturally occurring endophyte of various plants without causing any

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symptoms of disease;29 moreover, Ap has strong antagonistic activity against other

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microorganisms so is used in biological control of plant diseases.30 In necrotic (Nec) wood

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of the head and trunk, the presence of Fomitiporia mediterranea (Fm) and in rot (Rot)

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wood Fm and Pch was confirmed, which partially coincides with previous reports.1,4

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However, the presence of Pal and Fomitiporia punctata was not confirmed in any of the

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studied wood (Figure 3). Identified Fm sequences showed the highest similarity to

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sequences of this species from Italy, which was expected given that the grafts came from

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an Italian nursery; furthermore, the vineyard is situated a few kilometres from Italian

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border.

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After infection, for example by Phc, the fungus spread slowly through wood tissues,

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approx. 20-25 cm in nine months.31 Along the infection, the woody tissues, especially near

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the pathogen entrance, are first subject to discolouration, followed by necrosis and by

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rotting,1,32 which was also observed in our experiment. The discolouration is a

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consequence of phenolic alterations, usually caused by radical oxidative reactions induced

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by pathogens, leading to destruction of hemicelluloses and other wood fractions.32

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Individual phenolics. In total, irrespective of wood conditions and infections, 22

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different phenolics were identified, quantified and grouped according to chemical

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properties, gallic acid (GA), stilbenes (STB; 5 compounds), total flavanols (FLA) and

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monomeric flavanols (MON; 4 compounds), dimers (DIM; 8 compounds), trimers (TRIM;

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2 compounds) and tetramers (TETR; 2 compounds) of procyanidin together with

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derivatives (Table 1, Figure 2).

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In the group of hydroxybenzoic acids, only gallic acid (GA) was identified and

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quantified, the presence of which has already been mentioned in vine wood.1 The


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significantly highest GA (36.6 µg/kg) was measured in HLT wood of rootstock, and the

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lowest GA (14.5 µg/kg) in the head of Asym vines. In terms of Nec wood, the significantly

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highest GA content (76.7 µg/kg) was measured in the head of Asym vines (Table 2). The

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highest, 20-fold higher GA content, was observed in HLT wood of the head of Sym vines,

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while in the trunk and rootstock there was an approx. 6-fold higher content than in Asym

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vines. In Nec wood of Sym, in comparison to Asym vines and irrespective of the vine part,

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the GA contents were also significantly higher but only 1.8 to 4.0-fold. Moreover, the

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results also suggest that wood necrosis and rot on the head and trunk in Sym vines caused a

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decrease of GA; necrosis 1.5-2.1-fold and rot 11-21-fold in comparison to HLT wood,

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which is in accordance with previous reports.33 Lambert et al.10 did not find any inhibitory

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impact of GA on esca associated fungi and according to Mugnai et al.1, a “paradox” impact

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might occur – the abundance of GA in HLT wood increased in the vines in our study due

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to infection with Pch, but the higher GA content could enhance the growth of fungi by 15

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%.1

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In terms of number and their content in vine woods, flavanols (FLA) were the most

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abundant phenolic group. The highest share 76-86 % of total FLA content was provided by

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procyanidins (Pcys), followed by monomeric flavan-3-ols – catechin, epicatechin gallates

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and epicatechin (Table 2). Furthermore, of FLA oligomers, dimers (DIM) were the most

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abundant, followed by tetramers (TETR), monomers (MON) and trimers (TRIM) (Figure

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1). Esca infection significantly increased the content of total FLA, especially in HLT wood

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from the head and in Nec wood from the trunk and rootstock. We also observed that the

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different parts of the vine, irrespective of the wood conditions, responded to infection

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differently. For example, the total FLA content and most of their oligomers in HLT wood

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from the head and trunk of Asym vines were more or less equal but significantly higher



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than in rootstock (Figure 1), which might reflect the impact of different genotype.8,

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Furthermore, the contents of total MON, DIM and TRIM in Nec wood from the head of

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Asym vine showed a tendency to increase with necrosis, by approx. 1.1-2.0-fold, while it

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decreased by 1.3-2.0-fold in the trunk. The described differences suggest that annual and

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more intensive winter and summer pruning on the head than on the trunk causes more

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intense stress conditions, which might lead to FLA responses. In relation to Sym vines, the

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contents of total FLA and their oligomers in HLT wood from head were approx. 1.2-fold

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higher than in Nec wood, while in the trunk and rootstock, total FLA contents showed a

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tendency to increase with necrosis (Figure 1). In all samples of Rot wood, the content of

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total FLA and their oligomers was lower than in HLT and Nec woods, in which

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significantly the lowest total FLA contents were measured in Rot wood from the head – a

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7.8-10.6-fold decrease in comparison to HLT wood. In relation to Pcys, infection

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significantly increased their contents in all studied woods, irrespective of the vine part, 1.5

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to 4.1-fold in HLT and 1.3-3.6-fold in Nec wood. However, significantly the highest Pcys

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contents were in Nec wood from the trunk, followed by HLT wood from the head of Sym

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vines, in which also significantly the lowest Pcys contents in Nec and Rot wood were

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measured (Table 2). We also observed that Pcys contents, irrespective of vine part,

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significantly decreased with wood rotting (Table 2, Figure 1).

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The results suggest that the duration of the pathogen presence affected the FLA

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content. We speculate that, in the head of the vine, where the pathogen is present for a

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longer time than in the trunk and rootstock, the wood was subjected to necrosis for a longer

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period, causing stronger wood deteriorations, which caused a FLA decrease; however, Nec

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wood in the trunk and rootstock seems to be still partly active and FLA are still abundant.

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Moreover, a higher degree of FLA polymerisation could be a response of the wood to the


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presence of the pathogen but during wood necrosis the content of FLA oligomers

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decreased. Destruction of wood fractions leads to oligomer release, which are highly

266

reactive with fungi proteins34 – bonds cause less soluble compounds, which are difficult to

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extract and identify.

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Catechin (Cat) was the most abundant (119-1890 µg/kg) of the group of monomer

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flavan-3-ols (MON), irrespective of vine and wood conditions, followed by epicatechin

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gallates (Epi-Gs; 112-1156 µg/kg) and epicatechin (Epi; 19.9-436 µg/kg) (Table 2). The

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greatest impact of esca infection was observed in the content of Cat and Epi in Nec wood

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from the trunk reaching 7.8- and 5.2-fold significantly higher contents, respectively, which

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is approximately twice more than previously reported.35 On the other hand, necrosis also

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significantly increased the content of Cat, Epi and Epi-Gs in wood from the head of Asym

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but decreased it in Sym vines. A further reduction of their contents was observed in Rot

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wood, especially in the head of Sym vines, in which the contents of identified flavan-3-ols

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were 7-15-fold lower than in HLT wood. An opposite impact of necrosis was observed on

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the trunk and rootstock of Sym vines, in which the content of Cat, Epi and Epi-Gs

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significantly increased in wood from the trunk and rootstock, while in the trunk of Asym

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vines it significantly decreased (Table 2).

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In relationship to HLT wood from Sym vines, irrespective of vine part, the highest

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impact of infection was observed with Epi (2.8-fold), followed by Cat (2.4-fold) and Epi-

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Gs (2.3-fold), which are in the ranges of previous reports.35 Cat in HLT wood of Sym vines

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showed a tendency to decline from the head to rootstock, which was also observed with

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Epi and Epi-Gs, the contents of which did not differ in trunk and rootstock (Table 2) – that

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tendency might be explained by the different duration of the presence of the pathogen in



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various parts of the vine. Cat and Epi have no inhibitory effect on fungi Fm and Pch, but

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higher contents of Epi-Gs in woods might induce the colonisation of Pch.10

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The identified stilbenes (STB) were resveratrol glycosides (Res-Gly; 3 phenolics),

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astringin (Astrg) and ε-viniferin glucoside (Vini-Glu) (Table 2). As expected,14 esca

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infection, irrespective of vine part, increased the content of total STB in HLT wood 2.0-

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3.7-fold, especially in the head, where significantly the highest total STB, 260 µg/kg, was

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measured compared to the trunk and rootstock (Figure 1). An increase of total STB content

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was observed not only at the infection site but also in Nec wood from the head and trunk of

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Asym vines, 10- and 6.1-fold, respectively. Furthermore, the increase of total STB content

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in Nec wood from Sym, irrespective of vine part, was only from 3.7 to 4.3-fold higher than

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in HLT wood. In relation to Rot wood, the content of total STB was significantly lower in

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the head than in the trunk, but both were lower than in Nec wood (Figure 1). The obtained

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results in our study in relation to total STB content were much higher than previously

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reported data16 but, at the same time, STB responses to esca infection reflected three times

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higher STB contents in brown-red than in Asym wood. In terms of individual identified

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and quantified stilbenes, irrespective of vine status and part, Res-Gly were the most

303

abundant, followed by Vini-Glu and Astrg as the lowest (Table 2). In HLT wood of Asym

304

vines, irrespective of the vine part, the content of Res-Gly was 131-152 µg/kg, of Vini-Glu

305

3.33-9.78 µg/kg and of Astrg between 1.58 to 3.11 µg/kg, which increased approx. 6.4-,

306

5.1- and 7.8-fold, respectively, with necrosis. In general, esca infection significantly

307

increased the content of all identified stilbenes, especially in HLT wood from the head, in

308

which the content of Vini-Glu increased 5.3-fold, of Res-Gly 3.7-fold and of Astrg 3.6-

309

fold, which partly coincides with previous findings.16-17 However, ε-Vini-Gluerin and

310

astringin showed a significant inhibitory effect on the growth of some Botryosphaeria sp.,


Page 15 of 32


15

311

while trans-resveratrol only on Eutypa lata.10 In relation to Nec wood of Sym vines, the

312

greatest impact of necrosis was observed in Astrg content, which significantly increased,

313

4.6-4.8-fold, followed by Res-Gly 2.9-4.6-fold, but in the rootstock, the content of

314

identified STBs increased in general only 1.3-1.8-fold (Table 2), which might be explained

315

by different genotype.33 The identified STBs showed a tendency to decline in Rot

316

compared to Nec wood, approx. 7.4-fold in the head and only 2.0-fold in the trunk, in

317

which their contents also remained higher than in HLT wood, in which they were not

318

observed in the head – suggesting that STB accumulation depends on the duration and

319

species of fungi presence and their impact on wood degradation and conditions.1, 32, 34

320

Total phenolic contents. Generally speaking, significantly higher contents of total

321

analysed phenols (TAP) were measured in Sym vines than in Asym vines, especially in

322

Nec wood of the trunk (16364 µg/kg), followed by rootstock (14478 µg/kg) and head

323

(12970 µg/kg) (Figure 4). In HLT wood, a phenolic response to esca infection was also

324

observed, in which a significantly, 1.5-2.4-fold, higher TAP content was measured than in

325

Asym vines - the highest content 14383 µg/kg, which coincides with previous reports,17

326

was measured in the head, followed by the trunk and rootstock. In relation to HLT wood

327

from Asym vines, the TAP contents did not differ between head and trunk, but both

328

differed in comparison to rootstock, which could be attributed to the genotype.33 Moreover,

329

TAP contents in wood of the head of Asym vines showed a tendency to increase further

330

due to necrosis (1.7-fold higher). However, an opposite effect was observed in Sym vines,

331

confirming previous findings in relation to oxidative pressure.32, 36 It can be speculated that

332

the main reason for the decrease of TAP content in the head of Sym vines can be attributed

333

to the formation of tannin-protein complexes after the binding of fungi derived enzymes

334

with tannins. Tannin-protein complexes are insoluble and cannot therefore be analysed by



Page 16 of 32

16

335

the standard phenolic extraction procedure.34 An opposite effect on TAP content was

336

observed in the trunk and rootstock of Sym vines, in which TAP contents increased with

337

necrosis 1.6- and 2.8-fold, respectively. In any case, significantly the lowest TAP content

338

(1409 µg/kg) was observed in Rot wood, especially in the head of Sym vines (Figure 4), in

339

which the presence of wood deterioration, in comparison to the trunk and rootstock, was

340

advanced. The obtained results regarding the TAP in Rot wood suggested that the wood

341

rotting leads to a general phenolics decline, possibly by oxidation.

342

We demonstrated for the first time that the phenolics response to infection by esca

343

associated fungi differed according to the wood conditions and vine part. In general, esca

344

associated fungi significantly increased the phenolics content in all studied wood,

345

especially in necrotic wood of the trunk and rootstock, while the lowest increase was

346

observed in rotten wood in the head. The response of esca-free vines on wood necrosis in

347

the head was observed as an increase of phenolics, while those infected reduced the

348

phenolics content with necrosis. In the trunk and rootstock of infected vines, an opposite

349

effect was observed, whereby the phenolics contents increased with necrosis. The highest

350

decrease in phenolics content was demonstrated in rotten wood, especially in the head of

351

infected vines, in which wood deterioration was more advanced than in the trunk and

352

rootstock. The data also suggested that a higher degree of procyanidins polymerisation

353

might be a significant response to infection. However, the obtained results showed that

354

esca infection caused different responses of phenolics in different parts of the vine,

355

apparently gradually along the longitudinal spread of the pathogens through the vine. The

356

findings might suggest that phenolics alterations reflect not only the presence but also the

357

duration of the presence of the fungi colonizing the vine from the head, through the trunk

358

to the rootstock.


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17

359



Page 18 of 32

18

360

ABBREVIATIONS

361

Asym asymptomatic (esca)

362

Sym

symptomatic (esca)

363

HLT

healthy (wood)

364

Nec

necrotic (wood)

365

Rot

rotten (wood)

366

Fm

Fomitiporia mediterranea

367

Pch

Phaeomoniella chlamydospora

368

Pal

Phaeoacremonium aleophilum

369

Ap

Aureobasidium pullulans

370

GA

gallic acid

371

STB

stilbenes

372

FLA

flavanols

373

Pcys

procyanidins

374

MON monomers (Pcys)

375

DIM

376

TRIM trimers (Pcys)

377

TETR tetramers (Pcys)

378

TAP

total analysed phenols

379

Cat

catechin

380

Epi

epicatechin

381

Epi-Gs epicatechin gallates

382

Res-Gly

383

Astrg astringin

384

Vini-Glu ε-viniferin glucoside

dimmers (Pcys)

resveratrol glycosides

385 386

ACKNOWLEDGEMENT


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This work is part of the programme Horticulture No. P4-0013-0481 only, funded by the

388

Slovenian Research Agency. The study is of great interests for the COST FA 1303 action

389

Sustainable control of grapevine trunk diseases.

390 391

REFERENCES

392 393

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394 395

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Gramaje, D.; García-Jiménez, J.; Armengol, J., Grapevine rootstock susceptibility to

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Plant Pathol. 2014, 63, 185-192.

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antioxidant capacity in strawberries. J. Agric. Food Chem. 2002, 50, 6534-6542.

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from affected leaves and twigs of cherry laurel trees. Fol. Oecol. 2011, 38, 137-145.

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33. Bruno, G.; Sparapano, L., Effects of three esca-associated fungi on Vitis vinifera L.:

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I. Characterization of secondary metabolites in culture media and host responses to

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36. Close, D. C.; McArthur, C., Rethinking the role of many plant phenolics - protection from photodamage not herbivores? Oikos 2002, 99, 166-172.

501



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502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543

Figures Figure 1. Average content of total stilbenes (STB), flavanols (FLA) and their monomers (MON), dimers (DIM), trimers (TRIM) and tetramers (TETR) with standard errors (means±S.E., mg/L) according to the infection (Asym–vines without symptoms; Sym– symptomatic vines) and wood conditions (HLT–healthy wood, Nec–necrosis, Rot–rotten) in different parts (head, trunk and rootstock) of the ‘Cabernet Sauvignon’ variety. Lowercase letters (a, b…) indicate significant differences between Asym and Sym vines in identical vine parts and wood conditions, while uppercase letters (A, B…) stand for significant differences among the particular vine part at the identical wood condition respective of Asym and Sym vines; determined by the Duncan test (p < 0.05). Figure 2. HPLC chromatograms of the identified phenolics according to the wood conditions (HLT–healthy wood, Nec–necrotic wood, Rot–rotten wood) in the head of ‘Cabernet Sauvignon’ variety. a Number of an identified phenolic compound refers to the Table 1. Figure 3. Molecular Phylogenetic analysis of fungal sequences obtained from grapevine wood by Maximum Likelihood method. The tree with the highest log likelihood (3178.1133) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 32 nucleotide sequences. All positions containing gaps and missing data were eliminated. HLT Asym – Healthy wood from asymptomatic vines HLT Sym – Healthy wood from symptomatic vines Nec Sym – Necrotic wood from symptomatic vines Rot Sym – Rotten wood from symptomatic vines ITA – Italy, GER – Germany, EST – Estonia, FIN – Finland, ESP – Spain Figure 4. Average content of total analysed phenolics (TAP) with standard errors (means±S.E., µg/kg) according to the infection (Asym–vine without symptoms; Sym– symptomatic vines) and wood conditions (HLT–healthy wood, Nec–necrosis, Rot–rotten) in different parts of ‘Cabernet Sauvignon’ variety. Lowercase letters (a, b…) indicate significant differences in HLT wood, uppercase letters (A, B…) in Nec wood and numbers (1, 2) in Rot wood among particular vine parts and irrespective of the infection; determined by the Duncan test (p < 0.05).


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25

544 545

Table 1. Identification of phenolic compounds in grapevine wood in negative ions with HPLC-MS and MS2 Peak No. 1 2 3 4 5 6 7 7 8 9 10 11 11 12 13 14 15 16 17 17 18 19 19 20 21

[M-H](m/z) 169 577 577 865 577 289 865 1153 577 441 577 289 745 729 881 471 1153 435 441 577 905 435 405 435 615

MS2 (m/z) 125 425, 407, 289 407, 425, 289 695, 577, 425, 407, 289 425, 407, 289 245 695, 577, 407, 425, 289 865, 577, 451, 407, 425, 289 425, 407, 289 395, 289, 169 407, 425, 289 245 559, 407, 577, 619, 289 577, 559, 451, 407, 289 729, 559, 407, 169 377, 349, 255 865, 577, 451, 407, 425, 289 389, 227 289, 331, 169, 271 425, 407, 289 811, 799, 649, 451, 359 389, 227 243 389, 227 453, 359

Phenolic compound Gallic acid Procyanidin dimer 1 Procyanidin dimer 2 Procyanidin trimer 1 Procyanidin dimer 3 Catechin Procyanidin trimer 2 Procyanidin tetramer 1 Procyanidin dimer 4 Unidentified peak 1 Procyanidin dimer 5 Epicatechin (Epi)catechin-epigallocatechin gallate 2 Monogalloylated procyanidin dimer 1 Digalloylated procyanidin dimer Unidentified peak 2 Procyanidin tetramer 2 Resveratrol glycoside 1 Epicatechin gallate Procyanidin dimer 6 Unidentified peak 3 Resveratrol glycoside 3 Astringin Resveratrol glycoside 4 ε-viniferin glucoside

546 547

Legend:

548

Peak No. - Peak number



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26

Table 2. Phenolics Identified and Quantified (µg/kg FW) in Grapevine Wood of ‘Cabernet Sauvignon’ according to the Esca Infection Phenols

Gallic acid Procyanidinse Catechin Epicatechin Epi-Gsf Res-Glyg Astringin Vini-Gluh

Asym HLT 14.5±0.6a ab, Ac 4947±171 a, B 596±25 a, B 93.2±2.5 a, B 198±24 a, A 152±17 a 3.11±0.30 a, B 8.25±0.71 a, B

Head Nec 76.7±8.5 a, B 6706±903 B 826±89 a, B 171±29 a, B 804±118 B 1435±25 a, B 32.9±0.5 a, B 58.5±4.0 a, B

Rot -d -

HLT 20.8±0.5 a, AB 5475±274 a, B 525±19 a, B 104±3 a, B 424±44 a, B 131±14 a 2.56±0.10 a, AB 9.78±0.48 a, B

Trunk Nec 21.1±0.2 a, A 3364±722 a, A 242±89 a, A 56.4±2.7 a, A 275±20 a, A 453±17 a, A 13.2±2.9 a, A 30.6±1.7 a, A

Rot -

Rootstock HLT Nec 36.6±4.0 a, B 1855±100 a, A 182±22 a, A 75±4 A, a 242± 46 a, A 149±17 a 1.58±0.70 a, A 3.33±0.19 a, A -

Rot -

Sym Head Trunk Rootstock HLT Nec Rot HLT Nec Rot HLT Nec Rot Gallic acid 301±10 b, C 140±17 b, A 14.2±0.4 124±5 b, A 84.2±14.7 b, A 10.9±2.7 223±32 b, B 265±26 B Procyanidins 11291±229 b, B 8794±111 A 871±3 A 8351±114 b, A 12168±285 b, B 2112±187 B 7588±462 b, A 10359±762 AB Catechin 1815±32 b, C 1051±35 b, A 119±3 A 1144±23 b, B 1890±54 b, B 201±18 B 358±33 b, A 1713±55 B Epicatechin 311±24 b, B 235±28 b, A 19.9±1.6 A 197±3 b, A 294±35 b, A 68.3±3.4 B 236±40 b, A 436±23 B Epi-Gs 788±21 b, B 658±73 A 112±5 A 540±23 b, A 819±92 b, AB 258±15 B 587±79 b, A 1156±56 B Res-Gly 542±15 b, C 1593±76 b, C 250±18 A 235±17 b, A 1090±131 b, B 509±29 B 311±35 b, B 515±31 A Astringin 11.4±0.4 b, B 55.1±2.0 b, B 5.57±0.21 A 5.26±0.15 b, A 24.0±3.0 b, A 12.2±0.8 B 7.75±0.90 b, A 9.83±0.6 A Vini-Glu 43.5±2.6 b, B 102±10 b, B 17.5±0.2 A 13.9±0.3 b, A 57.7±2.6 b, A 29.8±1.7 B 13.1±0.8 b, A 23.5±0.4 A Abbreviations: FW–fresh weight; Asym–non symptomatic vines; Sym–symptomatic vines; vine parts (Head–upper part of the trunk, Trunk–central part of the trunk, Rootstock–vine under graft); wood conditions (HLT–healthy wood, Nec–necrotic wood, Rot–rotten wood); Epi–epicatechin; Glu–glucoside; Gs–gallates; Gly–glycoside; Res–resveratrol; Vini–viniferin; a The average content with standard error (mean±S.E.). b Lowercase letters indicate significant differences between Asym and Sym vines in identical vine parts and wood conditions; determined by the Duncan test (p < 0.05). c Uppercase letters indicate significant differences among the particular vine part at the identical wood condition respective of Asym and Sym vines; determined by the Duncan test (p < 0.05). d Not identified. e Sum of all identified procyanidins (dimers, trimers and tetramers) refers to Table 1. f Sum of all identified epicatechin gallates refers to Table 1. g Sum of all identified resveratrol glycosides refers to Table 1. h ε-viniferin glucoside refers to Table 1.

26 ACS Paragon Plus Environment

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ROT

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HLT

Figure 2. HPLC chromatograms of the identified phenolics according to the wood conditions (HLT–healthy wood, Nec–necrotic wood, Rot–rotten wood) in the head of ‘Cabernet Sauvignon’ variety. a Number of an identified phenolic compound refers to the Table 1. ACS Paragon Plus Environment

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Figure 3. Molecular Phylogenetic analysis of fungal sequences obtained from grapevine wood by Maximum Likelihood method. The tree with the highest log likelihood (-3178.1133) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 32 nucleotide sequences. All positions containing gaps and missing data were eliminated. HLT Asym – Healthy wood from asymptomatic vines HLT Sym – Healthy wood from symptomatic vines Nec Sym – Necrotic wood from symptomatic vines Rot Sym – Rotten wood from symptomatic vines ITA – Italy, GER – Germany, EST – Estonia, FIN – Finland, ESP – Spain


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Figure 4. Average content of total analysed phenolics (TAP) with standard errors (means±s.e., μg/kg) according to the infection (Asym–vine without symptoms; Sym–symptomatic vines) and wood conditions (HLT–healthy wood, Nec–necrosis, Rot–rotten) in different parts of ‘Cabernet Sauvignon’ variety. Lowercase letters (a, b…) indicate significant differences in HLT wood, uppercase letters (A, B…) in Nec wood and numbers (1, 2) in Rot wood among particular vine parts and irrespective of the infection; determined by the Duncan test (p < 0.05).



TOC graphic 86x49mm (300 x 300 DPI)


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Phenolic Responses to Esca-Associated Fungi in Differently Decayed

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